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Shigeki Kiyonaka

Publications and source records attributed to Shigeki Kiyonaka.

10 recordsLinked to original sources
PubMed2006

Oligo-Asp tag/Zn(II) complex probe as a new pair for labeling and fluorescence imaging of proteins.

To accomplish the selective labeling of a specific protein in complicated biological systems, a peptide tag incorporated into the protein and a complementary small molecular probe are required. Although a variety of peptide tag/probe pairs have been developed as molecular tools for protein analyses, the availability of pairs suitable for real-time imaging of proteins is still limited. We now report a new peptide tag/artificial probe pair composed of a genetically encodable oligo-aspartate sequence (D4 tag, (D4)n, n = 1-3) and the corresponding multinuclear Zn(II) complexes (Zn(II)-DpaTyrs). The strong binding affinity of the Zn(II)-DpaTyr probes with the D4 tag was a result of the multiple coordination bonds and the multivalent effect. It was measured quantitatively by isothermal titration calorimetry. The high affinity between the tag and the probe, indispensable for the selective protein labeling, enabled the pair to be used for the labeling and fluorescence imaging of a membrane-bound receptor protein tethering a triply repeated D4 tag ((D4)3) in an intact cell configuration without significantly affecting the receptor signal transduction.

Amino Acid Sequence↗
PubMed2006

Blocker-resistant presynaptic voltage-dependent Ca2+ channels underlying glutamate release in mice nucleus tractus solitarii.

The visceral sensory information from the internal organs is conveyed via the vagus and glossopharyngeal primary afferent fibers and transmitted to the second-order neurons in the nucleus of the solitary tract (NTS). The glutamate release from the solitary tract (TS) axons to the second-order NTS neurons remains even in the presence of toxins that block N- and P/Q-type voltage-dependent Ca(2+) channels (VDCCs). The presynaptic VDCC playing the major role at this synapse remains unidentified. To address this issue, we examined two hypotheses in this study. First, we examined whether the remaining large component occurs through activation of a omega-conotoxin GVIA (omega-CgTX)-insensitive variant of N-type VDCC by using the mice genetically lacking its pore-forming subunit alpha(1B). Second, we examined whether R-type VDCCs are involved in transmitter release at the TS-NTS synapse. The EPSCs evoked by stimulation of the TS were recorded in medullary slices from young mice. omega-Agatoxin IVA (omega-AgaIVA; 200 nM) did not significantly affect the EPSC amplitude in the mice genetically lacking N-type VDCC. SNX-482 (500 nM) and Ni(2+) (100 microM) did not significantly reduce EPSC amplitude in ICR mice. These results indicate that, unlike in most of the brain synapses identified to date, the largest part of the glutamate release at the TS-NTS synapse in mice occurs through activation of non-L, non-P/Q, non-R, non-T and non-N (including its posttranslational variants) VDCCs at least according to their pharmacological properties identified to date.

Analysis of Variance↗
PubMed2005

Three distinct read-out modes for enzyme activity can operate in a semi-wet supramolecular hydrogel.

Assays of hydrolytic enzyme activity, such as of glycosidases and phosphatase, as well as several proteases, using a semi-wet supramolecular hydrogel array composed of a glycosylated amino acetate are described. It has been demonstrated that the microcavity formed by gel fibrils is suitable to immobilize native enzymes without denaturation under semi-wet conditions, and thus the nanofiber has been rationally used as a sensing domain to monitor enzymatic reactions. By using a fluorogenic substrate, reducing the size of the hydrogel can significantly improve the problem of suppressed diffusion within the gel matrix thus making the hydrogel a promising semi-wet matrix for evaluating enzyme activity. Confocal laser scanning microscopy observations have shown that an environmentally sensitive fluorescent probe accumulates in the hydrophobic domain of the gel fiber and emits fluorescence more strongly upon hydrolytic cleavage of the substrate peptides. Not only a simple environmentally sensitive probe but also a FRET (fluorescence resonance energy transfer)-type read-out mode can be devised to analyze the enzymatic hydrolysis-triggered redistribution of the probe between the nanospace and the nanofiber to accomplish a more clearly distinguished enzyme assay. Thus, it is clear that three distinct read-out modes, that is, 1) fluorogenic substrates, 2) substrates bearing an environmentally sensitive probe, or 3) a substrate exhibiting FRET, can operate under the semi-wet hydrogel conditions used in these investigations. In addition, owing to the unique properties of the present supramolecular hydrogel in semi-wet conditions, that is, its phase-segregation properties and dynamics, the supramolecular substrate/enzyme array has successfully been used for high-throughput screening of single and multiple enzymes based on their activity, lysate analysis, and quantitative evaluation of inhibitor potency and selectivity.

Enzyme Inhibitors↗
PubMed2005

pH-Responsive shrinkage/swelling of a supramolecular hydrogel composed of two small amphiphilic molecules.

A pH-responsive volume-change function was successfully introduced into a supramolecular hydrogel that contained GalNAc-appended (GalNAc=N-acetylgalactosamine) glutamate ester 1 by the simple mixing of it with an appropriate amount of 2 a or 2 b amphiphilic carboxylic acid. In the 1:1 mixture (1:2), the hydrogel swelled under neutral pH conditions, but shrank to almost half of its original volume under acidic pH conditions. The structure and pH response of the mixed hydrogel were characterized by using X-ray diffraction (XRD), confocal laser scanning microscopy (CLSM), transmission or scanning electron microscopy (TEM, SEM), and Fourier transform IR (FTIR) spectroscopy. Well-developed fibers formed a stable hydrogel by self-assembly, and under acidic conditions the charge of the carboxylic acid terminal (from the carboxylate anion) was neutralized and then these fibers became densely packed. This macroscopic pH response was also applied to the pH-triggered release of bioactive substances. In this mixed supramolecular hydrogel, the hydrogelator 1 provides a stable hydrogel structure and the additive 2 acts as a commander that is sensitive to an environmental pH signal. The present supramolecular copolymerization strategy should be useful for the construction of novel, stimuli-responsive, soft materials.

Journal Article↗
PubMed2004

Activation of RasGRP3 by phosphorylation of Thr-133 is required for B cell receptor-mediated Ras activation.

The Ras signaling pathway plays a critical role in B lymphocyte development and activation, but its activation mechanism has not been well understood. At least one mode of Ras regulation in B cells involves a Ras-guanyl nucleotide exchange factor, RasGRP3. We demonstrate here that RasGRP3 undergoes phosphorylation at Thr-133 upon B cell receptor cross-linking, thereby resulting in its activation. Deletion of phospholipase C-gamma2 or pharmacological interference with conventional PKCs resulted in marked reduction in both Thr-133 phosphorylation and Ras activation. Moreover, mutation of Thr-133 in RasGRP3 alone severely impaired its ability to activate Ras in B cell receptor signaling. Hence, our data suggest that PKC, after being activated by diacylglycerol, phosphorylates RasGRP3, thereby contributing to its full activation.

Amino Acid Sequence↗
PubMed2003

Semi-wet peptide/protein array using supramolecular hydrogel.

The protein microarray is a crucial biomaterial for the rapid and high-throughput assay of many biological events where proteins are involved. In contrast to the DNA microarray, it has not been sufficiently established because of protein instability under the conventional dry conditions. Here we report a novel semi-wet peptide/protein microarray using a supramolecular hydrogel composed of glycosylated amino acetate. The spontaneous gel-formation and amphiphilic properties of this supramolecular hydrogel have been applied to a new type of peptide/protein gel array that is compatible with enzyme assays. Aqueous cavities created in the gel matrix are a suitable semi-wet reaction medium for enzymes, whereas the hydrophobic domains of the fibre are useful as a unique site for monitoring the reaction. This array system overcomes several drawbacks of conventional protein chips, and thus can have potential applications in pharmaceutical research and diagnosis.

Equipment Design↗
PubMed2003

Combinatorial library of low molecular-weight organo- and hydrogelators based on glycosylated amino acid derivatives by solid-phase synthesis.

A combinatorial approach for the synthesis of supramolecular gelators as new organic materials is described herein. In the course of the development of a convenient and flexible solid-phase synthesis of the artificial glycolipids, some of these compounds were accidentally found to act as low molecular-weight gelators toward organic solvents. Using this combinatorial solid-phase synthesis of glycosylated amino acetates, screening and optimization of low molecular-weight organo/hydro-gelators were efficiently carried out. We found that an N-acetyl-galactosamine-appended amino acid ester (GalNAc-aa) efficiently gelates a broad spectrum of organic solvents. More interestingly, some GalNAc-aa derivatives displayed an excellent hydrogelation capability. Transmission electron microscopy, scanning electron microscopy, confocal laser scanning microscopy, and FT-IR were used for characterization of the gel structure. It is indicated that supramolecular fibers supported by strong hydrogen-bonding networks are entangled so that the resulting spaces can immobilize a number of solvent molecules effectively. In addition, the supramolecular hydrogel consisting of GalNAc-suc-glu(O-methyl-cyc-pentyl)(2) is stable even under high salt concentrations probably due to its nonionic character and as a result, a native protein is successfully entrapped in the gel matrix without denaturation.

Amino Acids↗
PubMed2002

Site-specific discrimination by cyanovirin-N for alpha-linked trisaccharides comprising the three arms of Man(8) and Man(9).

Cyanovirin-N (CVN) is a novel cyanobacterial protein that selectively binds with nanomolar affinities the mammalian oligosaccharides Man(8) and Man(9). Consequently, CVN potently blocks HIV entry through highly avid carbohydrate-mediated interactions with the HIV-envelope glycoprotein gp120, and is under preclinical investigation as an anti-HIV microbicide. CVN contains two non-overlapping carbohydrate-binding sites that bind the disaccharide Manalpha(1-2)Manalpha (which represents the terminal disaccharide of all three arms of Man(9)) with low to sub-micromolar affinities. The solution structure of a 1:2 CVN:Manalpha(1-2)Manalpha complex revealed that CVN recognizes the stacked conformation of Manalpha(1-2)Manalpha through a deep hydrophilic-binding pocket on one side of the protein (site 2) and a semi-circular cleft on the other (site 1). With the prominent exception of the C1 hydroxyl group of the reducing mannopyranose ring, the bound disaccharide is positioned so that each hydroxyl group is involved in a direct or water-mediated hydrogen bond to the polar or charged side-chains comprising the binding pocket. Thus, to determine whether the next-most reducing mannopyranose ring will augment CVN affinity and selectivity, we have characterized by NMR and ITC the binding of CVN to three synthetic trisaccharides representing the full-length D1, D2 and D3 arms of mammalian oligomannosides. Our findings demonstrate that site 1 is able to discriminate between the three related trisaccharides methyl Manalpha(1-2)Manalpha(1-2)Man, methyl Manalpha(1-2)Manalpha(1-3)Man and methyl Manalpha(1-2)Manalpha(1-6)Man with remarkable selectivity, and binds these trisaccharides with K(A) values ranging from 8.1x10(3)M(-1) to 6.6x10(6)M(-1). Site 2 is less selective in that it binds all three trisaccharides with similar K(A) values ranging from 1.7 to 3.7(+/-0.3)x10(5)M(-1), but overall binds these trimannosides with higher affinities than site 1. The diversity of pathogenic organisms that display alpha(1-2)-linked mannosides on their cell surfaces suggests a broad defensive role for CVN in its cyanobacterial source.

Amino Acid Sequence↗
PubMed2002

First thermally responsive supramolecular polymer based on glycosylated amino acid.

In materials science, a dynamic property sensitive to an environmental change (heat, light, electric current, pH, and other chemical or physical changes) is indispensable for intelligent materials. Such organic materials, however, are very limited even in conventional polymers. This paper clearly demonstrates that, regardless of the low molecular weight, a glycosylated amino acid derivative newly screened by a combinatorial method forms a macroscopic supramolecular hydrogel that reversibly swells or shrinks in response to the external temperature. Using the unique thermal response of the present hydrogel, we carried out the controlled release of DNA and the perfect removal of bisphenol A from the polluted water. Recently, advanced supramolecular polymers, in which monomers are noncovalently connected, are expected to be highly advantageous over traditional polymers because of their tunable and recyclable characteristics. The present result newly confers a dynamic feature on the supramolecular polymers, which is desirable for the sophisticated application in many fields.

Acetylgalactosamine↗